WNK (With No lysine (K)) protein kinases are Ste20 relatives that we discovered through studying control of mitogen-activated protein kinases (MAPKs). WNKs are ubiquitous multipurpose, multifunctional enzymes that have been found in screens to identify proteins required for development and survival, and that affect growth in flies, worms, and mammals. WNK1 is essential for embryonic development in mice. The best known function of WNK family members is their influence on ion balance. We have identified a dozen WNK1 targets that include proteins that directly activate ion transporters; regulate endocytosis, exocytosis and vesicular trafficking; and are components of signal transduction pathways essential for growth and development. In this application, we propose experiments to elucidate mechanisms of WNK action and the functions of selected WNK target molecules. The Ste20 protein kinase oxidative stress responsive 1 (OSR1) and its homolog the serine, proline, alanine-rich kinase (SPAK) bind to WNK1 in cells, are WNK1 substrates, and are activated by WNK1 phosphorylation. The stimulus-dependent regulation of their association, specificity of their WNK binding, and the ion cotransporters and signal transduction pathways they regulate will be determined. We will also elucidate mechanisms of regulation of the epithelial sodium channel (ENaC) by WNK1. The serum- and glucocorticoid-inducible protein kinase SGK1 phosphorylates the E3 ubiquitin ligase neural precursor cell expressed, developmentally down regulated 4-2 (Nedd4-2). Nedd4-2 induces down regulation of a number of membrane proteins including ENaC; this results in reduced sodium reabsorption. Phosphorylation by SGK1 interferes with ENaC downregulation by Nedd4-2. We found that SGK1 and ENaC are regulated by WNK1. The underlying molecular mechanisms remain unknown. We will examine mechanisms of SGK1 regulation and the effects of WNK1 directly on Nedd4-2. Finally, we will explore the actions of WNK1 on vesicular trafficking. WNK1 is localized on numerous small cytoplasmic vesicles and increases the amount of transforming growth factor (TGF) 2 receptors and other proteins on the plasma membrane. We will characterize effects of WNK1 on the endocytosis and exocytosis of membrane proteins and analyze protein complexes associated with WNK1 vesicles. PUBLIC HEALTH RELEVANCE WNKs are required for development and survival, and affect growth control in organisms ranging from worms to man. Mutations in either WNK1 or WNK4 cause pseudohypoaldosteronism type II (PHA II), a form of high blood pressure caused by mutation of a single gene. Understanding the mechanisms of action of this family will provide significant insights into the control of ion balance and other essential processes, and may reveal novel strategies to develop anti-hypertensive therapies.